An anatomy of a multicellular organism retains a harmonious function as a whole by each cell taking a separate role. Otherwise, when part of the cells becomes cancerous (hereinafter referred to as a cancer, including tumors), the part grows into a neoplasm different from its peripheral region. However, the cancerous region and a normal tissue region away therefrom may not necessarily be sorted by a certain borderline, that is, the region surrounding the cancer is affected in some way. In order to analyze a function of an organ tissue, therefore, it is necessary to sort a small number of cells present in a small region.
Otherwise, in the medical field, in order to examine a region suspected of a cancer in the normal tissue, it is necessary to sort the region suspected of the cancer from a piece of tissue sorted in biopsy. For separation of such specific cells, it is common to fix the cells, perform various cell staining, and cut out a target part. A method called laser microdissection has been recently developed for correcting target cells only in a region subjected to the laser.
Otherwise, in the field of regeneration medicine, there is an endeavor to sort a stem cell from the tissue, cultivate the stem cell, and conduct the differentiation induction to regenerate the target tissue, and furthermore an organ.
To classify, identify or sort cells, it is necessary to distinguish the different cells according to a certain reference. Common methods of distinguishing cells include the following:
1) Visual cell classification based on morphology: an examination for a bladder cancer, an urethral cancer and the like by detection of an atypical cell present in urine, and a cancer screening by a classification of the atypical cells in blood or a cytological diagnosis in the tissue can be taken as examples.
2) Cell classification based on the cell surface antigen (marker) staining by the fluorescent antibody test: this is to stain a cell surface antigen, generally called as a CD marker, with a fluorescent labeling antibody specific thereto, and used for cancer screenings by a cell sorting using a cell sorter, a flow cytometer, or tissue staining. These techniques are frequently used not only in the medical field but also for the cytophysiological study and the industrial use of the cells.
3) Separation of a stem cell involves an example of purifying a differentiated target stem cell from roughly separated stem cells using a fluorescent pigment taken into a cell as a reporter and by actually re-cultivating the differentiated stem cell afterward. That is to say, since an effective marker for the stem cell has not yet been established, the target cell is selected by their differentiated characteristics of cells after their cultivation.
Separating and retrieving a specific cell in a culture fluid in this way is an important technique for biological and medical analyses. When cells are sorted based on a difference in the specific gravity of the cells, the cells can be sorted by the velocity sedimentation method. However, when there is little difference in the specific gravity of the cells enough to differentiate a non-sensitized cell from a sensitized cell, it is necessary to sort the cells one by one based on information from staining with the fluorescent antibody or other visual information. This technique may be represented by, for instance, a cell sorter. The cell sorter employs the technique as follows: The cells after the fluorescent staining processing are dropped into a charged droplet as isolated in the unit of cell, and a high electric field is irradiated in any direction on the plane perpendicular to the dropping direction in the process of the droplet dropping, whereby the dropping direction of the droplet is controlled by the irradiated voltage, based on the optical measurement of the presence and localization of the fluorescence in the cell in the droplet and the intensity of the light scattering diffraction, to fractionate and retrieve the droplet in a plurality of containers placed at the bottom (non-patent document 1: Kamarck, M. E., Methods Enzymol. Vol. 151, p 150-165 (1987)).
However, this technique involves the following problems: the cost is high; the system is large; a high electric field of some thousand volts is required; a large amount of samples is required; cells may be damaged during generation of the droplets; the sample cannot be directly observed. To solve these problems, a cell sorter has been recently developed which generates fine flow paths using the micromachining technology and sorts the cells flowing through the laminar flow in the flow path while directly observing them under a microscope (non-patent document 2: Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998); Analytical Chemistry, 70, pp. 1909-1915 (1998)). However, since the cell sorter which generates the fine flow paths using the microfabrication technology is slow in the response speed of the sample sorting with respect to the observation unit, another processing method that does not damage the sample and is faster in response is required in order to put the cell sorter into practical use.
In order to solve the problems, the present inventors have filed the applications for a cell analyzer/sorter capable of fractionating the samples based on the fine optical image of the sample and the distribution and localization of the fluorescence in the sample utilizing the microfabrication technology and easily analyzing/sorting the sample cells without damaging the samples retrieved (JP-A 2003-107099, JP-A 2004-85323, WO2004/101731). This apparatus is a substantially useful cell sorter for use in a laboratory, but for practical industrial/medical use, new techniques are required for the microfluidic pathway, cell transportation, retrieving method, and sample preparation.    [Non-Patent Document 1]
Kamarck, M. E., Methods Enzymol. Vol. 151, p 150-165 (1987)    [Non-Patent Document 2]
Micro Total Analysis, 98, pp. 77-80 (Kluwer Academic Publishers, 1998); Analytical Chemistry, 70, pp. 1909-1915 (1998)    [Patent Document 1]
JP-A 2003-107099    [Patent Document 2]
JP-A 2004-85323    [Patent Document 3]
WO2004/101731